Programmed cell death plays critical roles in a wide variety of physiological processes, including fetal development, tissue homeostasis, immune cell education, and elimination or virus- infected cells. Defects in the regulation of cell suicide mechanisms contribute to the pathogenesis of multiple diseases, including those characterized by insufficient (cancer; autoimmunity) and excessive cell death (stroke, myocardial infarction, AIDS). CED-4 family proteins are central regulators of apoptosis, which bind to and trigger the activation of caspase-family cell death proteases. These proteins contain an ATP-binding protein oligomerization domain (NB-ARC) coupled with domains that bind the pro-forms of particular caspases. The focus of this proposal is the biochemical and biological characterization of novel mammalian CED-4-like proteins, recently discovered by our research team: NAC, CARD-X, and DAP-3. NAC contains an NB-ARC domain together with a Caspase-Associated Recruitment Domain (CARD). CARD-X is another CARD-containing protein, which lacks a NB-ARC domain thus disqualifying it from CED-4-family membership, but which is a candidate regulator of caspase-activation pathways controlled by these proteins. DAP-3 is a regulator of apoptosis pathways induced by Tumor Necrosis Factor (TNF)-family death receptors, which contains a NB-ARC-like domain and a Death Effector Domain (DED)-like region that binds DED-containing pro-caspases. The goals of this proposal are to elucidate in full-detail the mechanisms by which these proteins control apoptosis pathways and to explore their in vivo roles using gene knock-out approaches in mice. The information provided by these investigations may provide insights into human diseases where dysregulation of programmed cell death is known to occur and may lead to new strategies for therapeutic intervention.